Method for enzymatic treatment of tissue products

ABSTRACT

Methods for treating tissue matrices and tissue matrices produced according to the methods are provided. The methods can include treating a tissue matrix with a proteolytic enzyme to produce a desired pliability of the tissue matrix and/or to control the immunogenicity of the tissue matrix. The methods can also comprise performing an assay to determine if contacting the at least one collagen-containing tissue matrix with a proteolytic enzyme has altered the at least one collagen-containing tissue matrix to reduce a human immune response to the tissue matrix. The methods can comprise treatment with alcalase under conditions controlled to produce a desired pliability without unacceptable alteration in collagen structure.

This application is a continuation-in-part under 35 U.S.C. §120 of U.S.application Ser. No. 14/019,274, which was filed on Sep. 5, 2013, andwhich is a continuation-in-part under 35 U.S.C. §120 of U.S. applicationSer. No. 13/457,791, which was filed on Apr. 27, 2012, and which claimspriority under 35 U.S.C. §119 to U.S. Provisional Application No.61/479,937, which was filed on Apr. 28, 2011, all of which are hereinincorporated by reference in their entirety.

The present disclosure relates to tissue matrices, and moreparticularly, to methods for controlling the mechanical and/orbiological properties of tissue matrices by treating the matrices withproteolytic enzymes.

Various tissue-derived products are used to regenerate, repair, orotherwise treat diseased or damaged tissues and organs. Such productscan include intact tissue grafts and/or acellular or reconstitutedacellular tissues (e.g., acellular tissue matrices from skin, intestine,or other tissues, with or without cell seeding). Such products generallyhave mechanical properties determined by the tissue source (i.e., tissuetype and animal from which it originated) and the processing parametersused to produce the tissue products. Since tissue products are oftenused for surgical applications and/or tissue replacement oraugmentation, the mechanical properties of the tissue products areimportant. For example, surgeons generally prefer tissues that feel likenatural tissues and/or are easy to handle during surgical procedures.Some tissue products, however, are undesirably stiff and/or have anunnatural feel. Accordingly, methods for treating tissue products toproduce more desirable mechanical properties are provided.

In addition, when implanted in the body, tissue products derived fromexogenous materials (e.g., tissues from other animals or patients, aswell as processed tissues of any type), may elicit an inflammatory orimmune response in the recipient. In some cases, an excessive immuneresponse may be detrimental, causing the implant to form undesirablescar tissue, or preventing suitable regeneration of tissue at the siteof implantation. Accordingly, methods for treating tissue products toreduce or control the immune response of the tissue products uponimplantation are provided.

SUMMARY

According to certain embodiments, a method for treating a tissue matrixis provided. The method can comprise selecting a collagen-containingtissue matrix and contacting the tissue matrix with a proteolytic enzymeunder conditions sufficient to produce a desired level of pliability inthe tissue matrix.

In another embodiment, a method for treating a tissue matrix isprovided. The method can comprise selecting a collagen-containingacellular tissue matrix and contacting the tissue matrix with aproteolytic enzyme under conditions sufficient to produce a desiredlevel of pliability in the tissue matrix and to increase the porosity ofthe tissue matrix.

In some embodiments, an acellular tissue matrix is provided. The matrixcan be prepared by a process comprising selecting an acellular tissuematrix and contacting the tissue matrix with a proteolytic enzyme underconditions sufficient to produce a desired level of pliability in thetissue matrix.

According to certain embodiments, a method for treating a tissue matrixis provided. The method can include selecting at least onecollagen-containing tissue matrix; contacting the at least onecollagen-containing tissue matrix with a proteolytic enzyme; andperforming an assay to determine if contacting the at least onecollagen-containing tissue matrix with the at least one proteolyticenzyme has altered the at least one collagen-containing tissue matrix toreduce a human immune response to the tissue matrix when the tissuematrix is implanted in a human body.

According to certain embodiments, a method for treating a tissue matrixis provided. The method can comprise selecting a dermal tissue matrix;and contacting the dermal tissue matrix with alcalase under conditionsincluding an incubation time in a solution having an alcalase activityselected to produce a desired increase in a pliability of the tissuematrix as measured by a drape test without producing significantcollagen degradation.

According to certain embodiments, a method for treating a tissue matrixis provided. The method can comprise selecting a dermal tissue matrix;and contacting the dermal tissue matrix with alcalase; and treating thetissue matrix with radiation, wherein the alcalase is provided underconditions selected to produce a desired tissue matrix pliability aftertreatment with radiation.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show acellular tissue matrices after treatment with enzymesusing methods described in Example 1, as well as untreated controls.

FIG. 2 is a box plot of tensile strength testing data for treated andcontrol samples, according to the methods of Example 1.

FIG. 3 is a box plot of suture strength testing data for treated andcontrol samples, according to the methods of Example 1.

FIG. 4 is a box plot of tear strength testing data for treated andcontrol samples, according to the methods of Example 1.

FIG. 5 is a box plot of elasticity testing data for treated and controlsamples, according to the methods of Example 1.

FIG. 6 is a box plot of creep resistance testing data for treated andcontrol samples, according to the methods of Example 1.

FIG. 7 illustrates DSC thermograms for untreated tissues and tissuestreated using bromelain and alcalase, according to Example 2.2.

FIGS. 8A-8B illustrate expression patterns of activation markers CD14(A) and CD163 (B) in monocytes co-cultured with various tissues, usingthe monocyte activation assay described in Example 2.4.

FIGS. 9A-F are hematoxylin & eosin (H&E) sections of untreated pADM(9A-9C) and enzyme treated pADM (9D-9F) after explant, as described inExample 2.5.

FIGS. 10A-D are H&E sections of untreated pADM (9A-9B) and enzymetreated pADM (9C-9D) explants, as describe in Example 2.5.

FIG. 11 is a bar graph showing drape test results for tissue matricesafter treatment with alcalase using methods described in Example 3, aswell as untreated controls.

FIG. 12 is a line graph showing collagenase digestion assay results fortissue matrices after treatment with alcalase using methods described inExample 3, as well as untreated controls.

FIG. 13 illustrates DSC results, including denaturation onsettemperature for untreated tissues and tissues treated using alcalaseaccording to Example 3.

FIG. 14 is an interval plot of maximum load/unit width for tissuematrices after treatment with alcalase using methods described inExample 3, as well as untreated controls.

FIG. 15 is an interval plot of elasticity for tissue matrices aftertreatment with alcalase using methods described in Example 3, as well asuntreated controls.

DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings. Wherever possible, the samereference numbers will be used throughout the drawings to refer to thesame or like parts.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Any range described herein will be understood toinclude the endpoints and all values between the endpoints.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

As used herein “tissue product” will refer to any human or animal tissuethat contains extracellular matrix proteins. “Tissue products” caninclude acellular or partially decellularized tissue matrices,decellularized tissue matrices that have been repopulated with exogenouscells, and/or cellular tissues.

As used herein, “tissue matrix” refers to the extracellular matrix,including collagen for a given tissue, wherein the matrix retains theinterconnected structural collagen matrix characteristics of the nativeextracellular matrix from the tissue from which it is derived.“Acellular tissue matrix” refers to a “tissue matrix” from which thenative tissue cells have been removed.

Various human and animal tissues can be used to produce products fortreating patients. For example, various tissue products forregeneration, repair, augmentation, reinforcement, and/or treatment ofhuman tissues that have been damaged or lost due to various diseasesand/or structural damage (e.g., from trauma, surgery, atrophy, and/orlong-term wear and degeneration) have been produced. Such products caninclude, for example, acellular tissue matrices, tissue allografts orxenografts, and/or reconstituted tissues (i.e., at least partiallydecellularized tissues that have been seeded with cells to produceviable materials).

For surgical applications, it is often desirable to produce tissueproducts that have certain mechanical properties. For example, thetissue product, which may include a sheet of material, should possesssufficient strength to withstand the demands of an intended use. Certaintissue products may be used to repair defects (e.g., hernias), tosupport surrounding tissues or implants (e.g., for breast augmentationand/or reconstruction), or to replace damaged or lost tissue (e.g.,after trauma or surgical resection). Whatever the particular use, thetissue product should have sufficient strength, elasticity, and/or othermechanical properties to function until tissue regeneration and/orrepair occurs.

In addition, tissue products should have a desirable feel. For example,surgeons generally prefer materials that have a natural tissue-like feel(e.g., are sufficiently soft, pliable, and/or elastic). Further, afterimplantation, it is desirable for tissue products to feel natural. Forexample, tissues used for breast augmentation should not be excessivelystiff so that upon implantation they produce a naturally feeling breast.

Some tissue products, however, can be excessively stiff for someapplications. For example, some surgeons note that porcine-deriveddermal materials such as STRATTICE™ are less pliable than human dermalproducts such as ALLODERM®. Processes for improving the feel of suchproducts, however, should not adversely affect the biological and/ormechanical properties of the products. Specifically, processing of theproducts to improve the feel of the products should not produce anundesirable decrease in other mechanical properties such as tensilestrength, and should not alter the protein matrix in such a way that thematerial does not support tissue regeneration and/or repair.

The present disclosure provides methods for treating tissues to controlthe feel of tissue products produced from the tissues. The disclosurealso provides tissue products produced using the methods of treatment.In addition, the present disclosure provides methods of treating tissuesto control the porosity of tissue products produced from the tissues. Insome cases, controlling the porosity can improve cellular infiltrationand tissue regeneration and/or repair.

In addition, the present disclosure provides methods for controlling orreducing an immune response to tissue matrices when implanted in a body.The immune response can be measured using a number of immunoassays,including monocyte activation assays, phagocytosis assays, and/oroxidative burst assays. The immunogenicity may also be controlled whiletreating the tissue to improve the feel of the tissue, to controlmechanical properties of the tissue (including any mechanical propertylisted herein), and/or to control porosity of the tissue. Aftertreatment of the tissue matrices, the matrices may be subjected to anassay to determine if the immunogenicity of the tissue has been alteredin a desirable way.

According to certain embodiments, a method for treating a tissue matrixis provided. The method can comprise selecting a dermal tissue matrix;and contacting the dermal tissue matrix with alcalase under conditionsincluding an incubation time in a solution having an alcalase activityselected to produce a desired increase in a pliability of the tissuematrix as measured by a drape test without producing significantcollagen degradation.

In certain embodiments, the enzymatic treatment conditions are selectedto provide a desired reduction in a drape value. As used herein, the“drape test” involves placing (draping) a sample of tissue matrix with aknown circular area on top of a circular pedestal having a knowndiameter. The projected area of the dermal matrix after it drapes overthe pedestal is divided by the original area of the tissue matrix circleto determine the drape value. Note that the area of the pedestal issubtracted from the original area of the disk and the draped area priorto dividing these two areas to determine drape. A lower value of drapemeans a softer (more drapable) material.

In certain embodiments, the enzyme is alcalase and the treatmentconditions are selected to produce a reduction in the drape value usinga 60 mm diameter tissue matrix sample and a 19 mm pedestal of between10-90%, between 20-80%, between 30-70%, between 40-60%, or about 50%(e.g., 45-55%), or at least 30%, at least 40%, at least 50%, at least60% or at least 70%. Further, in some embodiments, the conditions areselected to produce a drape value for a dermal acellular tissue ofbetween 0.2-0.4, or between 0.25-0.35, or less than 0.4, less than 0.35,less than 0.3, or less than 0.2.

A variety of factors may affect the final drape value. For example,factors that may be varied to control the effect on tissue pliabilitycan include, for example, temperature, pH, solution or buffer used,enzyme concentration, specific type of alcalase used, amount of solutioncompared to amount of tissue, and/or presence of inhibitors of enzymesin the solution.

As discussed in more detail below, alcalase has been found to have anumber of benefits in treatment of dermal tissue matrices. For example,at relatively low incubation times and enzymatic activities, alcalasehas been shown to provide a substantial increase in tissue pliabilityand improvement in tissue matrix drape tests (i.e., provides more drapecapability) without causing unacceptable alterations in collagenstructure or deterioration in other tissue properties such as tensilestrength, elasticity, tear strength, suture strength, creep resistance,burst strength, thermal transition temperature, collagenasesusceptibility or combinations thereof.

As discussed in detail in Example 3, treatment with alcalase followed byirradiation (e.g., as a terminal sterilization step) has been found tohave variable effects on tissue mechanical properties based onvariations in enzyme processing conditions. For example, with relativelylow enzyme concentrations and treatment times, followed by irradiation,the resulting tissue in an initial increase in tissue pliability, whichpeaks at a certain point, as shown, for example, in FIG. 11. Withfurther alcalase treatment (e.g., for longer treatment times or withhigher enzymatic activity), subsequent irradiation causes a loss ofpliability. Accordingly, in some embodiments, the methods can compriseselecting a dermal tissue matrix; and contacting the dermal tissuematrix with alcalase; and treating the tissue matrix with radiation,wherein the alcalase is provided under conditions selected to produce adesired tissue matrix pliability after treatment with radiation.

In order to maintain stability of the implanted extracellular matrix invivo, it is desirable to maintain the collagen stability duringprocessing. Collagen denaturation onset temperature by DSC and weightloss during treatment with collagenase enzyme are indicators of collagenstability. As used herein, “unacceptable degradation in tissue collagenstructure” means a change in DSC collagen denaturation onset temperatureof greater than 1° C., greater than 2° C., greater than 3° C., orgreater than 4° C., or greater than 5° C.

Various alacase activities and treatment times may be used. For example,alcalase may be provided in a solution with an activity of 1×10⁻⁶ Ansonunits per mL to 0.015 Anson units per mL, an activity of 1×10⁻⁶ units to1.5×10⁻³ Anson units per mL, or an activity of about 2×10⁻⁵ Anson unitsper mL to about 4×10⁻⁵ Anson units per mL. In addition, treatment timesmay vary between about 4 hours and 5 days.

In one embodiment, a method for treating a tissue matrix is provided.The method can comprise selecting a collagen-containing tissue matrixand contacting the tissue matrix with a proteolytic enzyme underconditions sufficient to produce a desired level of pliability in thetissue matrix. In another embodiment, a method for treating a tissuematrix is provided. The method can comprise selecting acollagen-containing acellular tissue matrix and contacting the tissuematrix with a proteolytic enzyme under conditions sufficient to producea desired level of pliability in the tissue matrix and to increase theporosity of the tissue matrix. FIGS. 1A-1D show acellular tissuematrices (STRATTICE™) after treatment with enzymes using methods of thepresent disclosure, as well as untreated controls. As shown, the treatedsamples are significantly more pliable that the untreated samples.

According to certain embodiments, a method for treating a tissue matrixis provided. The method can include selecting at least onecollagen-containing tissue matrix; contacting the at least onecollagen-containing tissue matrix with a proteolytic enzyme; andperforming an assay to determine if contacting the at least onecollagen-containing tissue matrix with the at least one proteolyticenzyme has altered the at least one collagen-containing tissue matrix toreduce a human immune response to the tissue matrix when the tissuematrix is implanted in a human body.

In various embodiments, treatment of tissue matrices with proteolyticenzymes provides improved mechanical properties without causingdegradation in one or more biological properties. For example, treatmentof tissue matrices can produce desired stiffness, feel, tactileproperties, and/or desired porosity without causing increasedinflammation or scar formation and/or without causing a reduction in thetissue matrices' ability to promote cell ingrowth and regeneration.

The tissue matrices can be selected to provide a variety of differentbiological and mechanical properties. For example, an acellular tissuematrix or other tissue product can be selected to allow tissue in-growthand remodeling to assist in regeneration of tissue normally found at thesite where the matrix is implanted. For example, an acellular tissuematrix, when implanted on or into fascia, may be selected to allowregeneration of the fascia without excessive fibrosis or scar formation.In certain embodiments, the tissue product can be formed from ALLODERM®or STRATTICE™, which are human and porcine acellular dermal matricesrespectively. Alternatively, other suitable acellular tissue matricescan be used, as described further below. The tissues can be selectedfrom a variety of tissue sources including skin (dermis or whole skin),fascia, pericardial tissue, dura, umbilical cord tissue, placentaltissue, cardiac valve tissue, ligament tissue, adipose tissue, tendontissue, arterial tissue, venous tissue, neural connective tissue,urinary bladder tissue, ureter tissue, and intestinal tissue. Themethods described herein can be used to process any collagenous tissuetype, and for any tissue matrix product. For example, a number ofbiological scaffold materials are described by Badylak et al., and themethods of the present disclosure can be used to treat those or othertissue products known in the art. Badylak et al., “Extracellular Matrixas a Biological Scaffold Material: Structure and Function,” ActaBiomaterialia (2008), doi:10.1016/j.actbio.2008.09.013.

In some cases, the tissue product can be provided as a decellularizedtissue matrix. Suitable acellular tissue matrices are described furtherbelow. In other cases, the method can further include processing intacttissue to remove cells or other materials. The tissues can be completelyor partially decellularized to yield acellular tissue matrices orextracellular tissue materials to be used for patients. For example,various tissues, such as skin, intestine, bone, cartilage, adiposetissue, nerve tissue (e.g., nerve fibers or dura), tendons, ligaments,or other tissues can be completely or partially decellularized toproduce tissue products useful for patients. In some cases, thesedecellularized products can be used without addition of exogenouscellular materials (e.g., stem cells). In certain cases, thesedecellularized products can be seeded with cells from autologous sourcesor other sources to facilitate treatment. Suitable processes forproducing acellular tissue matrices are described below.

A number of different enzymes can be used to treat the tissue matrices.For example, suitable enzymes can include sulfhydryl proteases such asbromelain. In addition, they can include bromelain, papain, ficin,actinidin, alcalase, trypsin or combinations thereof. The enzymes can bepurchased commercially or extracted from fruit sources. For example, onesource of bromelain is MCCORMICK MEAT TENDERIZER®, but the enzymes canalso be extracted from pineapple and/or purchased in a medical-gradeformulation.

The enzymes can be contacted with the tissues to increase the pliabilityof the tissue without causing undesirable degradation in othermechanical and/or biological properties. For example, when a batch ofmaterials are produced with or without the enzyme treatments discussedherein, the enzyme treatments will not produce an undesirable change inat least one of tensile strength, tear strength, suture strength, creepresistance, elasticity, collagenase susceptibility, burst strength,thermal transition temperature, or combinations thereof. In some cases,an undesirable change is a statistically significant reduction in anyone of tensile strength, tear strength, suture strength, creepresistance, glycosaminoglycan content, lectin content, burst strength,an increase in collagenase susceptibility or a change (upward ordownward) in thermal transition temperature (as measured usingdifferential scanning calorimetry).

As noted above, in some embodiments, the tissues are treated with anenzyme to increase the porosity of the tissue. In various embodiments,increasing the porosity of the tissue is performed to increase thenumber and/or size of channels, which can improve the rate of cellularinfiltration and tissue regeneration.

In some cases, the enzymes are selected such that they causesite-specific cleavage of proteins within the tissues. For example, ithas been found that treatment of porcine dermal materials with bromelaindoes not cause further alterations in the matrix structure after acertain amount of treatment. Therefore, treatment of dermis withbromelain does not cause further change in the matrix with prolongedexposure or after extended periods of time.

In addition, the enzyme can be applied to the tissues in a variety ofsuitable solutions. For example, bromelain has been found to beeffective when applied to tissues in normal saline, but other suitablebuffers (e.g., PBS) can be used.

As noted above, after treatment with an enzyme, an assay may beperformed to determine if contacting the at least onecollagen-containing tissue matrix with the at least one proteolyticenzyme has altered the at least one collagen-containing tissue matrix toreduce a human immune response to the tissue matrix when the tissuematrix is implanted in a human body. A number of suitable assays may beperformed. For example, suitable assays can include monocyte activationassays, phagocytosis assays, and oxidative burst assays.

In some embodiments, the assay may be performed on a segment or portionof the processed tissue, and other portions of the tissue may be used insubsequent medical or surgical procedures. In other embodiments, theassay may be performed on one or more samples from a batch of multiplesamples, and samples not subjected to the assay may be subsequentlyselected for use in treating a patient.

In certain embodiments, the enzyme treatment is selected to removecollagen or other proteins in the material that have a thermaltransition temperature that will permit denaturation at bodytemperature. For example, in some embodiments, dermal acellular tissuesare selected, and the enzymatic treatment is selected to remove athermal peak, as measured using DSC, between about 30 degrees and 40degrees Celsius. This small ECM peak is expected to denaturespontaneously at the human body temperature and may contribute to thedifferent inflammatory reactions that occur after implantation.

Acellular Tissue Matrices

The term “acellular tissue matrix,” as used herein, refers generally toany tissue matrix that is substantially free of cells and/or cellularcomponents. Skin, parts of skin (e.g., dermis), and other tissues suchas blood vessels, heart valves, fascia, cartilage, adipose tissue, bone,and nerve connective tissue may be used to create acellular matriceswithin the scope of the present disclosure. Acellular tissue matricescan be tested or evaluated to determine if they are substantially freeof cell and/or cellular components in a number of ways. For example,processed tissues can be inspected with light microscopy to determine ifcells (live or dead) and/or cellular components remain. In addition,certain assays can be used to identify the presence of cells or cellularcomponents. For example, DNA or other nucleic acid assays can be used toquantify remaining nuclear materials within the tissue matrices.Generally, the absence of remaining DNA or other nucleic acids will beindicative of complete decellularization (i.e., removal of cells and/orcellular components). Finally, other assays that identify cell-specificcomponents (e.g., surface antigens) can be used to determine if thetissue matrices are acellular. Skin, parts of skin (e.g., dermis), andother tissues such as blood vessels, heart valves, fascia, cartilage,bone, and nerve connective tissue may be used to create acellularmatrices within the scope of the present disclosure.

In general, the steps involved in the production of an acellular tissuematrix include harvesting the tissue from a donor (e.g., a human cadaveror animal source) and cell removal under conditions that preservebiological and structural function. In certain embodiments, the processincludes chemical treatment to stabilize the tissue and avoidbiochemical and structural degradation together with or before cellremoval. In various embodiments, the stabilizing solution arrests andprevents osmotic, hypoxic, autolytic, and proteolytic degradation,protects against microbial contamination, and reduces mechanical damagethat can occur with tissues that contain, for example, smooth musclecomponents (e.g., blood vessels). The stabilizing solution may containan appropriate buffer, one or more antioxidants, one or more oncoticagents, one or more antibiotics, one or more protease inhibitors, and/orone or more smooth muscle relaxants.

The tissue is then placed in a decellularization solution to removeviable cells (e.g., epithelial cells, endothelial cells, smooth musclecells, and fibroblasts) from the structural matrix without damaging thebiological and structural integrity of the collagen matrix. Thedecellularization solution may contain an appropriate buffer, salt, anantibiotic, one or more detergents, one or more agents to preventcross-linking, one or more protease inhibitors, and/or one or moreenzymes. In some embodiments, the tissue is incubated in thedecellularization solution overnight at 37° C. with gentle shaking at 90rpm. In certain embodiments, additional detergents may be used to removefat from the tissue sample.

After the decellularization process, the tissue sample is washedthoroughly. In some exemplary embodiments, e.g., when xenogenic materialis used, the decellularized tissue is then treated with adeoxyribonuclease (DNase) solution. Any suitable buffer can be used aslong as the buffer provides suitable DNase activity.

While an acellular tissue matrix may be made from one or moreindividuals of the same species as the recipient of the acellular tissuematrix graft, this is not necessarily the case. Thus, for example, anacellular tissue matrix may be made from porcine tissue and implanted ina human patient. Species that can serve as recipients of acellulartissue matrix and donors of tissues or organs for the production of theacellular tissue matrix include, without limitation, mammals, such ashumans, nonhuman primates (e.g., monkeys, baboons, or chimpanzees),pigs, cows, horses, goats, sheep, dogs, cats, rabbits, guinea pigs,gerbils, hamsters, rats, or mice.

Elimination of the α-gal epitopes from the collagen-containing materialmay diminish the immune response against the collagen-containingmaterial. The α-gal epitope is expressed in non-primate mammals and inNew World monkeys (monkeys of South America) as well as onmacromolecules such as proteoglycans of the extracellular components. U.Galili et al., J. Biol. Chem. 263: 17755 (1988). This epitope is absentin Old World primates (monkeys of Asia and Africa and apes) and humans,however. Id. Anti-gal antibodies are produced in humans and primates asa result of an immune response to α-gal epitope carbohydrate structureson gastrointestinal bacteria. U. Galili et al., Infect. Immun. 56: 1730(1988); R. M. Hamadeh et al., J. Clin. Invest. 89: 1223 (1992).

Since non-primate mammals (e.g., pigs) produce α-gal epitopes,xenotransplantation of collagen-containing material from these mammalsinto primates often results in rejection because of primate anti-Galantibody binding to these epitopes on the collagen-containing material.U. Galili et al., Immunology Today 14: 480 (1993); M. Sandrin et al.,Proc. Natl. Acad. Sci. USA 90: 11391 (1993); H. Good et al., Transplant.Proc. 24: 559 (1992); B. H. Collins et al., J. Immunol. 154: 5500(1995). Furthermore, xenotransplantation results in major activation ofthe immune system to produce increased amounts of high affinity anti-galantibodies. Accordingly, in some embodiments, when animals that produceα-gal epitopes are used as the tissue source, the substantialelimination of α-gal epitopes from cells and from extracellularcomponents of the collagen-containing material, and the prevention ofre-expression of cellular α-gal epitopes can diminish the immuneresponse against the collagen-containing material associated withanti-gal antibody binding to α-gal epitopes.

To remove α-gal epitopes, the tissue sample may be subjected to one ormore enzymatic treatments to remove certain immunogenic antigens, ifpresent in the sample. In some embodiments, the tissue sample may betreated with an α-galactosidase enzyme to eliminate α-gal epitopes ifpresent in the tissue. In some embodiments, the tissue sample is treatedwith α-galactosidase at a concentration of 300 U/L prepared in 100 mMphosphate buffer at pH 6.0. In other embodiments, the concentration ofα-galactosidase is increased to 400 U/L for adequate removal of theα-gal epitopes from the harvested tissue. Any suitable enzymeconcentration and buffer can be used as long as it is sufficient removalof antigens is achieved.

Alternatively, rather than treating the tissue with enzymes, animalsthat have been genetically modified to lack one or more antigenicepitopes may be selected as the tissue source. For example, animals(e.g., pigs) that have been genetically engineered to lack the terminalα-galactose moiety can be selected as the tissue source. Fordescriptions of appropriate animals see co-pending U.S. application Ser.No. 10/896,594 and U.S. Pat. No. 6,166,288, the disclosures of which areincorporated herein by reference in their entirety. In addition, certainexemplary methods of processing tissues to produce acellular matriceswith or without reduced amounts of or lacking alpha-1,3-galactosemoieties are described in Xu, Hui. et al., “A Porcine-Derived AcellularDermal Scaffold that Supports Soft Tissue Regeneration: Removal ofTerminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure,”Tissue Engineering, Vol. 15, 1-13 (2009), which is incorporated byreference in its entirety.

After the acellular tissue matrix is formed, histocompatible, viablecells may optionally be seeded in the acellular tissue matrix to producea graft that may be further remodeled by the host. In some embodiments,histocompatible viable cells may be added to the matrices by standard invitro cell co-culturing techniques prior to transplantation, or by invivo repopulation following transplantation. In vivo repopulation can beby the recipient's own cells migrating into the acellular tissue matrixor by infusing or injecting cells obtained from the recipient orhistocompatible cells from another donor into the acellular tissuematrix in situ. Various cell types can be used, including embryonic stemcells, adult stem cells (e.g. mesenchymal stem cells), and/or neuronalcells. In various embodiments, the cells can be directly applied to theinner portion of the acellular tissue matrix just before or afterimplantation. In certain embodiments, the cells can be placed within theacellular tissue matrix to be implanted, and cultured prior toimplantation.

Example 1 Treatment of Tissue Matrices to Increase Pliability

The following example illustrates a process for treating materialscomprising porcine dermal acellular tissue matrices with bromelain toincrease the pliability of the material. As discussed below, thetreatment did not cause an undesirable change in various mechanicalproperties. In addition, the treatment increases the porosity of thematerial, which may improve the rate of cellular infiltration and tissueregeneration.

For this experiment, STRATTICE™ acellular tissue matrices, as obtainedfrom LIFECELL CORPORATION (Branchburg, N.J.) were used. STRATTICE™ isavailable in a pliable form and a more firm version. Both types wereused for this experiment. The samples used for testing were cut intoquarters, and three quarters were treated. Untreated samples (1 quarter)were used as controls. The controls were refrigerated during treatment.STRATTICE™ is packaged in a solution, and therefore, does not requirerehydration. The treated samples were placed in 0.5 liters of cold tapwater containing 55 g of MCCORMICK MEAT TENDERIZER.

FIGS. 1A-1D show acellular tissue matrices after treatment with enzymesusing methods of the present disclosure, as well as untreated controls.FIGS. 2-6 are box plots of tensile strengths, suture strengths, tearstrengths, elasticity, and creep resistance for each treated and controlsamples. The treated samples had a noticeably increased pliabilitycompared to controls, but did not have significant reduction in othermechanical properties. In addition, no significant change in thermaltransition temperature or collagenase susceptibility was found. Overallpaired T-Test showed no statistical difference between control andtreatment groups.

Example 2 Treatment of Tissue Matrices to Modulate Immune Response UponImplantation

1. Preparation of Porcine Acellular Dermal Matrix (pADM)

Porcine skin was collected from an abattoir the epidermis andsubcutaneous fat were physically removed. The remaining dermal tissuewas de-contaminated at 37° C. in PBS containing antibiotics for 24hours.

Following de-contamination, the tissue was processed under asepticconditions. The dermal tissue was decellularized for 24 hours withdetergents to remove viable cells, washed with saline, and treated withDNAse/α-galactosidase or another 24 hours. Cellular debris and residualchemicals were removed by washing in PBS. The resulting porcineacellular dermal matrix (pADM) was stored at ambient temperature untiluse.

2. Preparation of Enzyme-Treated pADM

pADM was treated with one of two protease enzymes (alcalase orbromelain) overnight at 37° C. Bromelain, at a concentration of 100units/liter, was used to treat pADM either before the decellularizationor after the DNAse/α-galactosidase step. Alcalase was used at aconcentration of 0.003 Anson units/mL to treat pADM before thedecellularization step.

3. Differential Scanning Calorimetry of Treated Samples

The effect of bromelain enzyme treatment on porcine tissue ECM wasevaluated using differential scanning calorimetric (DSC) analysis.Tissue samples were hermetically sealed and scanned at 3° C./min from 2°C. to 120° C. DSC thermograms for untreated tissues and tissues treatedusing the enzymes (bromelain and alcalase) according to Example 2.2 areshown in FIG. 7. The thermograms demonstrated a few alterations in thetissue ECM after enzyme treatment. First, in contrast to decellularized,untreated control tissue, the enzyme-treated ECM did not show the smallpeak (˜2%) between 30° C. and 40° C. This small ECM peak is expected todenature spontaneously at the human body temperature and may contributeto different inflammatory responses elicited by untreated and enzymetreated pADM. Second, the two major ECM peaks above 55° C. weredepressed slightly by 0.9° C. on average. Third, the overall ECMdenaturation enthalpy was increased by ˜7.5% on average after enzymetreatment.

4. In Vitro Monocyte Activation

Monocytes are white blood cells that form part of the innate immunesystem. In response to inflammatory agents, they are rapidly activatedand initiate an inflammatory response. To predict human inflammatoryresponses to enzyme-treated and untreated tissues, monocytes wereisolated from human peripheral blood and incubated with the tissuesovernight. Following incubation, cells were washed and stained withantibodies against two surface markers used to monitor activation, CD14and CD163.

Upon activation, monocytes decrease expression of both CD14 and CD163 ontheir surfaces. This expression pattern was confirmed usinglipopolysaccharide (LPS), a known activator of monocytes. FIGS. 8A-8Billustrate expression patterns of activation markers CD14 (A) and CD163(B) in monocytes co-cultured with various tissues. The expression levelsof CD14 and CD163 in un-induced monocytes (black) serve as baselinenegative controls. For comparison, the expression patterns of thesemarkers in un-induced monocytes served as negative controls.Enzyme-treated pADM induced a much lower level of activation thanuntreated pADM, as evidenced by the expression pattern of both markers.In fact, the pattern and level of CD14 expression in monocytes exposedto enzyme-treated pADM are very similar to those of un-inducedmonocytes. Monocytes exposed to enzyme-treated pADM had slightlydecrease expression of CD163 but to a much lesser extent than monocytesexposed to either untreated pADM or LPS.

The results indicate that enzyme-treated pADM induces minimalinflammatory responses in human monocytes. Upon monocyte activation,expressions of both CD14 and CD163 decrease. This pattern is confirmedby cells co-cultured with a known monocyte activator, lipopolysaccharide(LPS). Untreated pADM (red) show lower expressions of CD14 and CD163compared to enzyme-treated pADM (green), indicating enzyme treatmentdecreases monocyte activation. Thus, enzyme-treated pADM elicit less ofan inflammatory response in human monocytes.

5. In Vivo Performance of pADM and Enzyme-Treated pADM

To assess the in vivo performance of pADM and enzyme-treated pADM, 1cm×1 cm pieces of pADM and enzyme treated pADM were implanted insubcutaneous tissue of immune competent rats. At two and four weeksafter implantation, the tissues were explanted and processed forhistological evaluation of inflammation, cellular repopulation, andrevascularization.

FIGS. 9A-F are hematoxylin & eosin (H&E) sections of untreated pADM(9A-9C) and enzyme treated pADM (9D-9F) after explant, as described inabove. FIGS. 10A-D are H&E section of untreated pADM (9A-9B) and enzymetreated pADM (C-D) explants, as described above. Enzyme treated pADMinduced minimal to no inflammation, while untreated pADM inducedmoderate to high inflammatory responses characterized by the presence ofabundant of immune cells. Furthermore, enzyme-treated pADM explantsexhibited enhanced cellular repopulation and revascularization comparedto untreated pADM. In the untreated pADM, fibroblast-like cells andvascular structures were present mainly on the periphery of the tissue.In contrast, those same cells and vascular structures were observedthroughout the enzyme treated pADM, including in the middle of thetissue.

6. Collagenase Digestion Assays

Different aliquots of freeze-dried pADMs and enzyme-treated (usingbromelain, trypsin, and alcalase) pADMs were weighed and digested withcollagenase Type I for varying lengths of time. At each time point, somealiquots of digested samples were separated from the collagenase Isolution by centrifugation and washed with water to remove residualcollagenase I solution. All aliquots were again freeze dried andweighed. The percentage of matrix remaining at each time point wascalculated by taking the ratio of the dry weight of the digested sampleto the dry weight of the initial sample.

Enzyme treatment did not negatively impact the susceptibility of pADMsto collagenase digestion. Both pADMs and enzyme treated pADMs weredigested by collagenase Type I to the same degree and at the same rate.Accordingly, the methods of the present disclosure have been found toprovide improved immunological properties upon implantation withoutcausing a degradation in collagenase susceptibility.

Example 3 Treatment of Tissues with Controlled Alcalase Activity

Porcine dermis was treated with alcalase having varying activity from3×10⁻⁵ to 0.015 Anson units/mL in an approximately neutral pH aqueoussolution for 16 hours. The porcine dermis was subsequentlydecellularized, disinfected, and sterilized with electron beam toprepare a sterile acellular dermal matrix. Various tests were performedto study the effect of alcalase on various mechanical, structural, andbiological properties.

FIG. 11 is a bar graph showing drape test results for tissue matricesafter treatment with alcalase using methods described in Example 3, aswell as untreated controls. The drape test involved placing a 60 mmdiameter circle of dermal matrix on top of a 19 mm diameter pedestal.The projected area of the dermal matrix after it drapes over thepedestal was divided by the original area of the 60 mm diameter circleto determine the drape value. Note that the area of the pedestal issubtracted from the original area of the disk and the draped area priorto dividing these two areas to determine drape. A lower value of drapemeans a softer (more drapable) material. The data shows that drapedecreases significantly (the material softens) for the lowest enzymeconcentration of 3×10⁻⁵ Anson units/mL. Above 3×10⁻⁵ Anson units/mL, atthe selected treatment time, temperature, and pH, the drape valueincreases and appears to plateau above 1.5×10⁻³ Anson units/mL.

FIG. 13 is a line graph showing collagenase digestion assay results fortissue matrices after treatment with alcalase using methods described inExample 3, as well as untreated controls. The data show similardigestion profile to the control (non-enzyme treated matrix) for thelower concentrations of alcalase with increasing rate of degradation forthe matrices exposed to higher concentrations of alcalase.

FIG. 14 illustrates DSC results, namely denaturation onset for untreatedtissues and tissues treated using alcalase, according to Example 3.Similar to collagen stability measured using collagenase susceptibilitytesting (FIG. 13), the lower denaturation onset with increasing alcalaseconcentrations indicates decreasing collagen stability as a function ofalcalase treatment concentration.

FIG. 15 is an interval plot of maximum load/unit width for tissuematrices after treatment with alcalase using methods described inExample 3, as well as untreated controls. This data was generated bytensile testing approximately 1 cm wide strips of the materials tofailure while measuring force. Testing was performed using and INSTRONmechanical test system. The data show similar strength for alltreatments.

FIG. 16 is an interval plot of elasticity for tissue matrices aftertreatment with alcalase using methods described in Example 3, as well asuntreated controls. Elasticity is a measure of stiffness of the materialat higher loads in the linear portion of the displacement-force curve.Elasticity is in contrast to the drape measurement, which reflectsstiffness at very low loads (the force of gravity on the tissue). Nosignificant changes in elasticity were seen between treatment groups.

The test shows that alcalase treatment effectively increases pliabilityof pADM with initial treatment, with subsequent increase in pliabilitywith continued treatment. Further, low levels of alcalase exposure donot adversely affect maximum load, collagenase susceptibility, andelasticity.

What is claimed is:
 1. A method for treating a tissue matrix,comprising: selecting a tissue matrix; and contacting the tissue matrixwith alcalase under conditions including an incubation time in asolution having an alcalase activity selected to produce a desiredincrease in a pliability measured by a drape test of the tissue matrixwithout producing significant collagen degradation.
 2. The method ofclaim 1, wherein the tissue matrix is contacted with the proteolyticenzyme under conditions that do not produce an undesirable change in atleast one of tensile strength, tear strength, suture strength, creepresistance, burst strength, thermal transition temperature, collagenasesusceptibility or combinations thereof.
 3. The method of claim 1,wherein the conditions are selected to provide a substantially maximumincrease in tissue pliability.
 4. The method of claim 1, wherein thetissue matrix is a non-primate tissue matrix.
 5. The method of claim 1,wherein the tissue matrix is a porcine tissue matrix.
 6. The method ofclaim 1, wherein the tissue matrix is contacted with the proteolyticenzyme under conditions that do not produce an undesirable change inelasticity.
 7. The method of claim 1, further including treating thetissue matrix to removal at least some of the cells and cellularcomponents from the tissue matrix.
 8. The method of claim 7, includingremoval of all the cells and cellular components from the tissue matrix.9. The method of claim 1, wherein the tissue matrix is an acellulartissue matrix.
 10. The method of claim 1, wherein the tissue matrixcomprises a dermal tissue matrix.
 11. The method of claim 1, whereincontacting the tissue matrix with alcalase includes contacting thetissue matrix with a solution having alcalase with an activity of 1×10⁻⁶units to 0.015 Anson units/mL.
 12. The method of claim 1, whereincontacting the tissue matrix with alcalase includes contacting thetissue matrix with a solution having alcalase with an activity of 1×10⁻⁶units to 1.5×10⁻³ Anson units/mL.
 13. The method of claim 1, whereincontacting the tissue matrix with alcalase includes contacting thetissue matrix with a solution having alcalase with an activity of about2×10⁻⁵ units to about 4×10⁻⁵ Anson units/mL.
 14. The method of claim 1,further comprising packaging the tissue matrix.
 15. The method of claim1, further comprising sterilizing the tissue matrix.
 16. The method ofclaim 1, wherein the tissue matrix is contacted with alcalase underconditions to produce reductions in the drape value of or at least 30%.17. The method of claim 1, wherein the tissue matrix is contacted withalcalase under conditions to produce reductions in the drape value of orat least 40%.
 18. The method of claim 1, wherein the tissue matrix iscontacted with alcalase under conditions to produce reductions in thedrape value of or at least 50%.
 19. The method of claim 1, wherein thetissue matrix is contacted with alcalase under conditions to producereductions in the drape value of or at least 60%.
 20. The method ofclaim 1, wherein the tissue matrix is contacted with alcalase underconditions to produce a drape value of 0.2-0.4.
 21. The method of claim1, wherein the drape test comprises placing a tissue matrix of a knowncircular area on top of a circular pedestal having a known diameter anddividing the projected area of the tissue matrix after it drapes overthe pedestal by the original area of the tissue matrix circle todetermine the drape value.
 22. An acellular tissue matrix made by themethod of claim
 1. 23. A tissue matrix, comprising: a porcine acellulartissue matrix having a drape value of less than 0.4.
 24. The tissuematrix of claim 23, wherein the drape value is less than 0.35.
 25. Thetissue matrix of claim 23, wherein the drape test comprises placing atissue matrix of a known circular area on top of a circular pedestalhaving a known diameter and dividing the projected area of the tissuematrix after it drapes over the pedestal by the original area of thetissue matrix circle to determine the drape value.
 26. A method fortreating a tissue matrix, comprising: selecting a dermal tissue matrix;and contacting the dermal tissue matrix with alcalase; and treating thetissue matrix with radiation, wherein the alcalase is provided underconditions selected to produce a desired tissue matrix pliability aftertreatment with radiation.
 27. The method of claim 26, wherein the tissuematrix is contacted with the proteolytic enzyme under conditions that donot produce an undesirable change in at least one of tensile strength,tear strength, suture strength, creep resistance, burst strength,thermal transition temperature, collagenase susceptibility orcombinations thereof.
 28. The method of claim 26, wherein the conditionsare selected to provide a substantially maximum increase in tissuepliability.
 29. The method of claim 26, wherein the tissue matrix is anon-primate matrix.
 30. The method of claim 26, wherein the tissuematrix is a porcine tissue matrix.
 31. The method of claim 26, furtherincluding treating the tissue matrix to removal at least some of thecells and cellular components from the tissue matrix.
 32. The method ofclaim 31, including removal of all the cells and cellular componentsfrom the tissue matrix.
 33. The method of claim 26, wherein the tissuematrix is an acellular tissue matrix.
 34. The method of claim 26,wherein the tissue pliability is measured using a drape test thatcomprises placing a tissue matrix of a known circular area on top of acircular pedestal having a known diameter and dividing the projectedarea of the tissue matrix after it drapes over the pedestal by theoriginal area of the tissue matrix circle to determine the drape value.35. The method of claim 34, wherein the tissue matrix is contacted withalcalase under conditions to produce reductions in the drape value of orat least 30%.
 36. The method of claim 34, wherein the tissue matrix iscontacted with alcalase under conditions to produce reductions in thedrape value of or at least 40%.
 37. The method of claim 34, wherein thetissue matrix is contacted with alcalase under conditions to producereductions in the drape value of or at least 50%.
 38. The method ofclaim 34, wherein the tissue matrix is contacted with alcalase underconditions to produce reductions in the drape value of or at least 60%.39. The method of claim 34, wherein the tissue matrix is contacted withalcalase under conditions to produce a drape value of 0.2-0.4.
 40. Themethod of claim 26, wherein the tissue matrix comprises a dermal tissuematrix.
 41. The method of claim 26, wherein the radiation comprisesE-beam radiation.
 42. The method of claim 26, wherein the radiationcomprises gamma radiation.